Measuring Picomolar Intracellular Exchangeable Zinc in PC-12 Cells Using a Ratiometric Fluorescence Biosensor Rebecca A. Bozym † , Richard B. Thompson †,* , Andrea K. Stoddard ‡ , and Carol A. Fierke ‡ † Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 21201, and ‡ Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109 Z inc is the second most abundant “trace” ele- ment in the body. This metal ion is vital for normal cellular function as a cofactor in numer- ous enzymes (1 ), in transcription factors (2, 3 ), in the immune system (4 ), and in the reproductive system (5 ). In the brain, synaptically released zinc has physi- ological and perhaps pathological relevance (6–9 ); the level of free zinc ions after release may reach a range of 10–100 μM in the synaptic cleft (10 ). Zinc has also been shown to modulate the response of NMDA recep- tors at nanomolar concentrations (11, 12 ). Although zinc is essential for proper brain function, zinc may also operate as a neurotoxin. Added zinc ions are toxic to neurons (13 ); furthermore, dying neurons fill with free zinc following prolonged seizure and ischemic insult (6, 14, 15 ). In addition, while zinc ions at various levels (up to hundreds of micromolar) induce apoptosis in some systems (16 ), the membrane-permeant chelator tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) also causes apoptosis, presumably due to excessive chela- tion of zinc. Finally, oxidative insults, including admin- istration of nitric oxide, lead to the release of free zinc from intracellular stores (17–20 ) including the release of bound zinc from metallothionein (20, 21 ). Although zinc plays important biological roles, little is known about the processes of distribution of this metal in the body or the incorporation of zinc into a variety of metalloproteins. Eukaryotic cells generally are rich in zinc with a total concentration in the range of 100 μM (1 ). However, the abundance of zinc ligands in cells, including metallothionein and other proteins, glutathione, histidine, cysteine, and diphosphate compounds (22, 23 ), ensures that the vast majority of cellular zinc is bound and not free. On the basis of the high affinity of certain zinc-sensitive transcription *To whom correspondence should be addressed. E-mail rthompso@umaryland.edu. Received for review December 30, 2005 and accepted February 12, 2006 Published online March 10, 2006 10.1021/cb500043a CCC: $33.50 © 2006 by American Chemical Society 103 www.acschemicalbiology.org VOL.1 NO.2 • ACS CHEMICAL BIOLOGY ABSTRACT Zinc plays both physiological and pathological roles in biology, making it of increasing interest. To date, intracellular free zinc has been measured in cell types supplemented with or enriched in zinc, such as hippocampal neurons. Here we quantitatively image intracellular exchangeable zinc in an ordinary resting cell culture line (PC-12), using an excitation ratiometric fluorescent biosensor based on carbonic anhydrase (CA). Human CA II has a K d of 4 pM for zinc and suffers no interference from millimolar calcium or magnesium ions. The CA-based biosensor was readily introduced into the cell by a novel approach: fusing a transactivator of transcription (TAT)-derived cell penetrating peptide to the CA molecule and adding it to the cells. Our results indicate that the resting concentration is approximately 5–10 pM in cytoplasm and nucleus. Interestingly, the tetrakis(2-pyridylmethyl)ethyl- enediamine (TPEN)–Zn complex and TPEN are both apoptogenic for this cell line.